PROJECT SUMMARY/ABSTRACT DESCRIPTION: See instructions. This must contain a summary of the proposed activity suitable for dissemination to the public (no proprietary/confidential information). It should be a self-contained description of the project and contain a statement of objectives and methods to be employed. It should be informative to other persons working in the same or related fields. DO NOT EXCEED THE SPACE PROVIDED. The goal of the proposed research is to discover biologically important genetic variants underlying risk for nicotine dependence (ND). We will identify genes with smoking-related differences in DNA methylation and RNA expression, map genetic variants underlying these differences, and conduct large-scale association testing of the regulatory genetic variants with ND. Cigarette smoking is the leading cause of preventable morbidity and mortality in the United States. Despite well-known health effects, 19.3% of U.S. adults are cigarette smokers. Genome-wide association study (GWAS) analyses of ND and other smoking phenotypes have conclusively identified associations with single nucleotide polymorphisms (SNPs) spanning nicotinic acetylcholine receptor and nicotine metabolizing genes. These SNPs have been translated downstream, showing effects on timing of cessation and lung cancer onset, as well as interaction with pharmacotherapy. In keeping with the generally observed enrichment of replicable SNP-disease associations from GWAS, we found that ND-associated SNPs include a functional missense SNP and several noncoding SNPs correlated with nearby DNA methylation (cis-methylation quantitative trait loci, cis-meQTLs) and RNA expression (cis-eQTLs). Despite these successes, much of the 50% heritability of ND is unexplained. We hypothesize that focusing on gene variants regulating methylation and expression in addiction-related brain regions will lead to novel discoveries. We propose the following: Specific Aim 1: Assess differences in human brain-specific DNA methylation and RNA expression levels between active smoking and nonsmoking decedents (deceased persons) Specific Aim 2: Perform QTL mapping to identify genetic variants that underlie the smoking-related differentially methylated and expressed genes. Specific Aim 3: Test eQTL and meQTL variants for association with ND Specific Aims 1 and 2 will use the biomarker cotinine to define smoking status and will leverage data from 240 post-mortem brains, focusing on two addiction-relevant regions: dorsolateral prefrontal cortex and nucleus accumbens. Specific Aim 3 will use an existing large-scale collection of GWAS samples defined by the Fagerstrm Test for ND: total N = 31,405 (23,556 European ancestry and 7,849 African American participants). Our integration of epigenomics in human brain and genomics of nicotine dependence will greatly improve the likelihood of meaningful discovery over standard GWAS by targeting gene regions and specific variants with high biological relevance specifically in the brain, while retaining a genome-wide scope.